Our long-term objectives are to understand the mechanisms of Na+ absorption in the human intestine. A family of nine members of the structurally related isoforms of Na+/H+ exchangers (NHE), named NHE1-9, has been identified. Of these, NHE1, 2 and 3 have been the most characterized isoforms and shown to be expressed in the mammalian intestine. The NHE1 is considered to be the housekeeping isoform and its encoded protein is localized to the basolateral membrane of the polarized epithelial cells. The NHE 2 and 3 isoforms are expressed in a tissue-restrictive manner and localized in the intestinal and kidney epithelial cells to the apical membrane. These isoforms are involved in vectorial Na+ absorption in the epithelial cells. Our studies emphasize that the NHE-2 isoform might be the predominant sodium-absorbing transporter in the colon. Recent reports of a compensatory mechanism for the lack of NHE2 in NHE2-null mice further support our hypothesis. We have cloned the human NHE2 cDNA and its promoter region and established that transcription factors, Sp1 and Sp3, play a major role in the basal and stimulated NHE2 promoter activity. Our recent findings show that NHE2 mRNA and promoter expression are reduced by proinflammatory cytokines, TNF-? and IFN-?. We hypothesize that the reduced level of NHE2 expression by these cytokines may lead to suppression of NHE activity coupled with the loss of absorption of electrolytes and water in diarrhea. Furthermore we have identified a suppressor element in the NHE2 5'-untranslated region (5'-UTR) that attenuates NHE2 promoter-driven reporter gene expression at the post-transcriptional level. Our recent studies also suggest that epigenetic mechanisms may be involved in tissue- specific expression of NHE2 gene. Therefore, it is critical to understand the mechanisms underlying NHE2 regulation both at the transcriptional and post-transcriptional levels and to identify factors mediating tissue- specific expression of this gene. As a continuation of our studies, we propose the following Specific Aims: 1) To define the molecular pathways leading to repression of NHE2 expression by proinflammatory cytokines TNF-? and IFN-?, 2) To investigate the mechanisms involved in post-transcriptional repression of NHE2 expression by 5'-UTR and 3) To study the effects of epigenetic regulation on the cell-specific expression of NHE2. Our proposed studies of the regulation of NHE-2 isoform may increase our understanding of the mechanisms of sodium absorption in the human colon and the potential role of this NHE isoform in pathophysiology of diarrheal disorders. PUBLIC HEALTH RELEVANCE: The mechanisms controlling electrolyte absorption by the mammalian intestine are relevant to the processes directly involved in the development of diarrhea related to infection, inflammation and other causes. Therefore, investigation of the mechanisms by which intestinal salt absorption could be altered is essential for clinical medicine. The project described in this submission focuses on one of the important transporter proteins functioning in sodium absorption by the human colon. Any information derived from studies of these proteins would be of clinical and therapeutic benefit for patients suffering from diarrheal disease processes.